The overall goal of this procedure is to demonstrate in vivo and in vitro techniques for the rearing of entero pathogenic nematodes. The in vivo method is accomplished by first incubating instar larvae of the greater wax moth garia melanoma with infective juvenile or IJ suspension. Next, the nematode infected cadavers are transferred to a modified white trap so that they can be collected.
The in vitro culture method is carried out by first repairing liver, kidney, or lipid agar plates, and the desired symbiotic bacteria are streaked out. Then IJ suspension is added to the plates and the nematodes are allowed to develop. Finally, the agar dish is transferred to a modified white trap for the harvesting of the nematodes.
Ultimately, these techniques successfully establish EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The inbivo and in vitro methods shown in this presentation can be used not only for research with pathogenic nematodes, but also for a wide array of experiments aimed at understanding ProCard and eary interactions. Demonstrating the procedures will be John McMullen graduate student in my laboratory.
To begin the in vivo rearing of Entero pathogenic nematodes, invert a 100 by 15 millimeter plastic Petri dish and place two discs of filter paper in the lid of the dish evenly distribute one milliliter of the infective juvenile or IJ suspension. On the filter paper, add 10 last in star larvae of the greater wax moth garia melanoma to the dish to create about 100 to 200 IJs pal lava. Cover the lid with the bottom of the Petri dish and label it.
Then place the dish inside a plastic bag, loosely seal it and incubated in the dark between 20 to 25 degrees Celsius. After three to five days, remove the cadavers with signs of nematode infection to a modified white trap to carry out the liver kidney method for the in vitro rearing of nematodes with their bacteria. Chop liver and kidney into small pieces and place in a blender with sodium chloride agar, and 300 milliliters of water blend until the meat becomes a liquid pulp, thick paste or puree.
Transfer the mixture to an Meyer flask and use 200 milliliters of water to rinse the blender vessel. Decanting the medium into the flask. Autoclave the mixture for 15 minutes at 121 degrees Celsius.
Pour approximately 20 milliliters of agar into five or six centimeter Petri dishes and allow the agar to solidify before storing the dishes at four degrees Celsius until needed. To prepare nematodes using the lipid agar method streak, the desired symbiotic bacteria onto a lipid agar plate, and incubate the plates in the dark between 20 to 25 degrees Celsius for 24 to 48 hours. The next day at approximately 0.4 milliliters of surface sterilized nematode suspension composed of either eggs or infective juveniles, and incubate the plates at room temperature in the dark or in an incubator at 23 plus or minus three degrees Celsius.
Monitor the cultures daily for the generation of IJs, which should occur in about 12 days. As soon as the nematodes are seen, crawling on the sides of the dish under a lamina flow hood. Harvest them by transferring the bottom of the dish with agar to a modified white trap.
When the IJs emerge from the white trap, collect them and use sterilized water to rinse the IJ suspension. To remove any medium debris debris, store the IJs in sterilized distilled water at 10 degrees Celsius in the cold, or use immediately using IJs at the desired nematode species. Infect 10 to 20 gella larvae or enough to produce the desired number of eggs.
After three or four days, collect the cadavers. Place each cadaver individually into a Petri dish with saline solution or M nine buffer. To dissect a cadaver, use fine tip forceps to pull on the head.
Use an L-shaped fine needle to collect at least 20 GRD females with eggs and place them in a watch glass containing 10 milliliters of M nine buffer. After preparing an ionizing solution according to the text protocol, use M nine buffer to rinse the females at least three times by filling the watch glass with buffer and carefully aspirating up the solution. Next, immediately add the ionizing solution and allow the females to remain in the solution for no more than 10 to 15 minutes.
To speed up the process of disintegration, use a needle or scalpel to cut the female bodies. Remove the eggs by pipetting them into micro centrifuge tubes. To further remove nematode tissue attached to the eggs, use a high speed setting to vortex the tubes for 15 seconds.
Alternatively, pipette the solution up and down several times After pelleting the eggs at approximately 18, 000 G for two minutes. Remove the ionizing solution before using sterile distilled water to rinse the eggs twice. Centrifuge your final time at 900 G for one minute.
Then suspend the eggs in 300 to 500 microliters of sterile distilled water and place them in a sterile three centimeter Petri dish or sterilized. Watch glass. Examine the eggs under a dissecting microscope at 30 to 50 x magnification to confirm that they are intact before transferring them to a five centimeter plate.
With liver kidney agar. Store the labeled plates upright in the dark at 28 degrees Celsius. The egg should hatch overnight, observe the dishes periodically and discard any dishes that show signs of fungal or bacterial contamination.
Once IJs are seen, migrating up the wall of the Petri dish, remove the lid and transfer the agar dish to a modified white trap. The in vivo rearing method uses live insects as hosts for nematode growth and reproduction. Shown here is an infection chamber with the last instar larvae of the greater wax moth garia Melan Ella.
This figure shows the in vitro rearing method using a lipid agar plate with mature female nematode stages. The presence of lipid droplets in the agar is very common, and they usually do not interfere with the nematodes growth or maturation. In this figure, IJs are seen crawling on the side of a liver kidney plate.
A disadvantage of this method is that it is prone to contamination with bacteria or fungi because of the medium's rich nature seen here as a liver kidney plate with Steiner ne nematodes in a modified white trap for the harvesting of IJs progeny. In this trap, the IJ stages of the nematodes will migrate to the water where they can be harvested for use in experiments or be stored until needed. For the rearing of APO symbiotic nematodes, it is recommended to grind the IJs and LB and plate the suspension onto NBTA medium to further confirm the absence of the bacterial symbiant.
Users should be aware that rating of pathogenic nematodes without their bacterial symbiance over multiple generations may have implications on nematodes survival and reproduction over time.
